Design and Synthesis of Polyheterocyclic Compounds Containing Pyrazolopyridopyrimidine Nucleus with Antimicrobial Activities

Abstract This study reports the design, synthesis, and antibacterial evaluation of a library of novel polyheterocyclic derivatives featuring a unique fused pyrimidopyridopyrazole moiety. A cyclocondensation reaction between an amino−pyrazolopyridopyrimidine precursor and malonates afforded a series of pyrimidopyridopyrazolopyrimidine derivatives. Further diversification was achieved through nucleophilic cyclocondensation, yielding a collection of complex polyheterocyclic systems encompassing various ring structures. All synthesized compounds were rigorously characterized using spectroscopic techniques and elemental analysis. The antibacterial activity of the newly synthesized compounds was assessed against a panel of Gram‐positive and Gram‐negative bacteria. Notably, several compounds exhibited promising antibacterial activity, highlighting their potential as leads for the development of novel antibiotics.


Introduction
Heterocyclic compounds and their nitrogen derivatives are esteemed for their diverse and potent biological and pharmacological properties, encompassing antimicrobial, antifungal, antimalarial, anti-inflammatory, analgesic, and potential anticancer activities. [1]Furthermore, Pyridine, a vital N-heteroaromatic structure, plays a central role in the design of therapeutic agents, displaying compelling biological effects.These pyridinebased heterocyclic compounds also serve as crucial intermediates in synthesizing a wide range of bioactive molecules. [2]They occur naturally in antibiotics, nucleic acid, hormones, vitamins, and other substances. [3]As a result, studies on synthesis are being conducted of poly-functionalized compounds, there has been a lot of interest in heterocyclic compounds that are crucial to the drug discovery process.Heterocycles with pyrazolo annulation, like pyrazolopyridopyrimidines, which have five nitrogen atoms in one molecule and three fused heterocyclic skeletons, may combine the characteristics of all three fused heterocycles and show a variety of uses in the textile and agricultural industries, [4] and the medical sector, such as antiproliferative agents. [5]These compounds exhibit a variety of biological traits, including virucidal, anticancer, fungicidal, bactericidal, and vasodilator activities. [6,7]Additionally, pyrazolopyridopyrimidine derivatives demonstrated strong antitumor effects and high antibacterial activities. [8]11] The results from the literature previously reported indicate that designing new chemical entities with increased activity may be facilitated by combining two or more bioactive heterocyclic pharmacophores into a single molecule.[19][20] Figure 2.
Motivated by the aforementioned findings, and in resumption of our prior projects [28][29][30] on the synthesis of new fusedpyrimidine scaffolds, we wanted to create new pyrazolo [4,3e]pyrido [1,2-a]pyrimidines compounds derivatives of fused with

Results and Discussion
We selected 5-chloro-1,3-dimethyl-2,4-dioxo-1,2,3,4tetrahydropyrido [2,3-d]pyrimidine-6-carbonitrile (1) which was prepared according to the reported method. [31]The choro derivative 1 was used to synthesis of the starting material after its treatment with hydrazine hydrate (80 %) to afford 3-amino-6,8-dimethyl-2H-pyrazolo [3',4':4,5]pyrido [2,3-d] The structure of the synthesized derivatives 3 a-e were confirmed using mass spectrometry and NMR.The structure of the starting derivative 2 was obviously confirmed by 1 H-NMR spectrum, which showed the presence of the single signals at δ 7.69 ppm, related to the NH 2 and NH of pyrazole ring respectively, as well as in the IR spectrum showed the disappearance of the characteristic peak related to CN group (2150 cm À 1 ), but the appearance of a single peaks at 3265 cm À 1 related to NH, NH 2 groups.The structure of derivatives 3 a-e were clearly confirmed by the spectral data. 1 H-NMR spectra of derivatives 3 a-e indicated the appearance of new single signals at δ 9.30 ppm and 10.12-13.12ppm for NH, and OH groups respectively of new pyrimidine ring formation, also, appearance of the new signals corresponding to proton (Ar), methyl, ethyl, n-butyl and/or phenyl groups at carbone-9.Besides, the MS spectrum revealed the molecular ion peaks at the exact molecular weight of the derivatives 3 a-e.

Biological Activity Assessment
In our investigation of novel antibacterial compounds (3a-e, 4, 5, 6a-d, 7, and 8), we employed a broth microdilution method to assess their efficacy against four prevalent bacterial strains.This method offers a quantitative measure of a compound's antibacterial activity by determining the minimum inhibitory concentration (MIC) and sub-MIC values. [32]The MIC represents the lowest concentration of the compound that completely inhibits visible bacterial growth after a defined incubation period. [33]Sub-MICs, on the other hand, represent concentrations below the MIC that still exert a measurable effect on bacterial growth. [34,35]e employed the following protocol to determine the MIC and sub-MIC values: 1) Serial dilutions of each compound were prepared in a cation-adjusted Mueller-Hinton broth (CAMHB) to achieve a broad range of concentrations.2) Standardized inocula of each bacterial strain were prepared and added to wells containing the diluted compounds.
3) The plates were incubated at 37°C for 24 hours (bacteria) to allow for bacterial growth.4) The MIC was determined as the lowest concentration of the compound that resulted in no visible bacterial turbidity compared to the growth control (bacteria with no test compound).5) Sub-MICs were determined by measuring the extent of bacterial growth inhibition at concentrations below the MIC.Ampicillin, a broad-spectrum antibiotic, was incorporated as a positive control in each assay.This ensures the validity of the assay by verifying that the bacteria are viable and susceptible to established antibiotics. [34]By comparing the activity of our test compounds to Ampicillin, we gain valuable context for interpreting their potency. [35,36]
Based on the sub-MICs in Table 1, compounds 6a-d, 4, and 8 emerged as the most potent candidates, exhibiting consistently lower values across various bacteria.This signifies their superior broad-spectrum antibacterial activity.
Pseudomonas aeruginosa: Compounds 8 and 4 displayed notable inhibition, with sub-MICs comparable to the reference antibiotic Ampicillin (Table 1).Conversely, compound 5 exhibited lower potency against this bacterium.
Staphylococcus pyogenes: Compounds 6a-d and 8 exhibited good activity, while compound 5 showed the highest inhibition (sub-MIC of 6.25 μg ml À 1 ) as shown in Figure 3.

Structure-Activity Relationship Study
The study reveals potent antibacterial activity within the tested compounds, with particular strength exhibited by 3 c, 3d, 4, 6ad, and 8.This efficacy can be traced back to the intricate molecular structures and functional groups present.Several key features likely contribute to their ability to combat bacteria: These aromatic moieties may interact with essential bacterial enzymes or receptors, disrupting crucial cellular processes.This complexity enhances the surface area available for binding to bacterial targets, potentially increasing potency.Substituents like phenyl, ethyl, and hydroxyethyl on the aromatic rings influence the molecule's interaction with bacterial components by modulating its hydrophobicity and hydrophilicity.In compounds 6a-d, the presence of a pyrrolidine ring and the unique pyrimidoÀ pyridoÀ pyrazolo core further elevate the molecular complexity, offering additional sites for interaction with bacterial targets.Additionally, the diverse substituents (methyl, ethyl, hydroxyethyl, and phenyl) contribute to a broader spectrum of antibacterial activity by tailoring the chemical structure to specific bacterial strains.Compound contain pyridazine ring adds another layer of structural intricacy.The methyl and phenyl substituents on this ring likely influence the compound's lipophilicity, impacting its ability to penetrate bacterial membranes.
Ultimately, the observed antibacterial activity arises from a confluence of factors.Functional groups like imino and hydroxyl groups facilitate hydrogen bonding with bacterial components, while the overall molecular architecture and the presence of polyheteroaromatic systems provide ample opportunities for targeted interactions.This interplay between structure and function, combined with the diversity within the tested compounds, leads to varying levels of potency against different bacterial strains, paving the way for more targeted and effective antibacterial strategies.

Experimental
All compounds were obtained commercially and were utilized without any further purifying.A Kofter block instrument was used to determine melting points (mp).TLC (silica gel 60F254, Merck, Darmstadt, Germany) monitored reactions. 1H and 13 C NMR spectra were measured on the standard apparatus (300 MHz, 75.5 MHz) using DMSO-d 6 or CDCl 3 to dissolve samples.A GC MS-QP 1000 EX Mass Spectrometer was used to obtain mass spectra (Shimadzu, Tokyo, Japan), Cairo University's Faculty of Science's Microanalytical Laboratory.Elemental analyses were carried out in the microanalysis laboratory at Cairo University using a Yanaca CHN Corder MT-3 elemental analyzer.The IR spectra have been captured using KBr disks on a Perkin-Elmer 1720 FT-IR spectrometer.Antibacterial potency was assessed at Prince Sattam Bin Abdul-Aziz University's College of Pharmacy, Department of Pharmaceutics.

Conclusions
Polyheterocyclic compounds containing four and five fused rings were prepared from the pyrazolo [3',4':4,5]pyrido [2,3d]pyrimidine as precursors.The preparation was accomplished by cyclocondensation reactions with malonates derivatives.But on the other hand, Dichloro derivatives undergo to nucleophilic cyclocondensation reactions, after its treatment with primary amines, phenyl hydrazine and/or thiourea, affording the polyherocyclic derivatives.The compounds have demonstrated potent antimicrobial activities against various microorganisms with sub-minimal inhibitory concentrations.This study highlights the importance of rational drug design and synthetic strategies in developing novel compounds with potential therapeutic applications.
[a] F. A. El-Essawy, M. A. A. Odah six and five membered heterocyclic rings with antibacterial activity testing.